This paper describes a biochemical study for making complexes between the nucleoprotein of influenza viruses A and B (A/NP and B/NP) and small RNAs (polyUC RNAs from 5 to 24 nucleotides (nt)), starting from monomeric proteins. We used negative stain electron microscopy, size exclusion chromatography-multi-angle laser light scattering (SEC-MALLS) analysis, and fluorescence anisotropy measurements to show how the NP-RNA complexes evolve. Both proteins make small oligomers with 24-nt RNAs, trimers for A/NP, and dimers, tetramers, and larger complexes for B/NP. With shorter RNAs, the affinities of NP are all in the same range at 50 mM NaCl, showing that the RNAs bind on the same site. The affinity of B/NP for a 24-nt RNA does not change with salt. However, the affinity of A/NP for a 24-nt RNA is lower at 150 and 300 mM NaCl, suggesting that the RNA binds to another site, either on the same protomer or on a neighbour protomer. For our fluorescence anisotropy experiments, we used 6-fluorescein amidite (FAM)-labelled RNAs. By using a (UC)6-FAM3&prime; RNA with 150 mM NaCl, we observed an interesting phenomenon that gives macromolecular complexes similar to the ribonucleoprotein particles purified from the viruses.

viruses-08-00247-f005: Ribonucleoprotein (RNP)-like species of influenza A virus nucleoprotein. Negative stain electron micrographs of A/NP with 5′phosphate-(UC)6-FAM3′. Complexes (ratio 1:1) were incubated at room temperature in 20 mM Tris-HCl pH 7.5, 150 mM NaCl, and 5 mM β-ME. Samples were negatively stained with sodium silicotungstate. (a) Kinetics of RNP-like formation with a 12-nucleotide RNA. Electron micrographs were taken at different times after incubation with several RNAs at room temperature. With an unlabelled RNA, no worm-like structure is observed, even overnight; (b) Worm-like structures after purification by centrifugation (10 min, 11,000× g, room temperature). The length of these species is random and they appear flexible. Similar to RNPs, these entities seem to be helical; (c) RNP-like species obtained with different RNA 5′phosphate-polyUC-FAM3′ lengths. With a 10-nt RNA, only a few short worm-like structures are observed. With 11 nt the worm-like structures are longer. With 12- and 13-nt RNAs, the worms are very similar with fewer trimers/tetramers in the background. The scale bars correspond to 100 nm. ON: overnight.

Mentions:
In order to assess the influence of both salt and RNA size on the binding of monomeric NP, a series of salt conditions (50, 150 and 300 mM NaCl) were tested against size-incremented polyUC RNA from 5 to 24 nt. The experiments were performed using SEC-MALLS (Figure 3 and Figure S2a,b), negative staining EM (Figure 4 and Figure 5) and fluorescence anisotropy (Figure 6 and Figure S4). A 5′phosphate-polyUC-OH3′ was used for the MALLS experiments and 5′phosphate-polyUC-FAM3′ for the negative staining EM and for the fluorescence anisotropy experiments.

viruses-08-00247-f005: Ribonucleoprotein (RNP)-like species of influenza A virus nucleoprotein. Negative stain electron micrographs of A/NP with 5′phosphate-(UC)6-FAM3′. Complexes (ratio 1:1) were incubated at room temperature in 20 mM Tris-HCl pH 7.5, 150 mM NaCl, and 5 mM β-ME. Samples were negatively stained with sodium silicotungstate. (a) Kinetics of RNP-like formation with a 12-nucleotide RNA. Electron micrographs were taken at different times after incubation with several RNAs at room temperature. With an unlabelled RNA, no worm-like structure is observed, even overnight; (b) Worm-like structures after purification by centrifugation (10 min, 11,000× g, room temperature). The length of these species is random and they appear flexible. Similar to RNPs, these entities seem to be helical; (c) RNP-like species obtained with different RNA 5′phosphate-polyUC-FAM3′ lengths. With a 10-nt RNA, only a few short worm-like structures are observed. With 11 nt the worm-like structures are longer. With 12- and 13-nt RNAs, the worms are very similar with fewer trimers/tetramers in the background. The scale bars correspond to 100 nm. ON: overnight.

Mentions:
In order to assess the influence of both salt and RNA size on the binding of monomeric NP, a series of salt conditions (50, 150 and 300 mM NaCl) were tested against size-incremented polyUC RNA from 5 to 24 nt. The experiments were performed using SEC-MALLS (Figure 3 and Figure S2a,b), negative staining EM (Figure 4 and Figure 5) and fluorescence anisotropy (Figure 6 and Figure S4). A 5′phosphate-polyUC-OH3′ was used for the MALLS experiments and 5′phosphate-polyUC-FAM3′ for the negative staining EM and for the fluorescence anisotropy experiments.

This paper describes a biochemical study for making complexes between the nucleoprotein of influenza viruses A and B (A/NP and B/NP) and small RNAs (polyUC RNAs from 5 to 24 nucleotides (nt)), starting from monomeric proteins. We used negative stain electron microscopy, size exclusion chromatography-multi-angle laser light scattering (SEC-MALLS) analysis, and fluorescence anisotropy measurements to show how the NP-RNA complexes evolve. Both proteins make small oligomers with 24-nt RNAs, trimers for A/NP, and dimers, tetramers, and larger complexes for B/NP. With shorter RNAs, the affinities of NP are all in the same range at 50 mM NaCl, showing that the RNAs bind on the same site. The affinity of B/NP for a 24-nt RNA does not change with salt. However, the affinity of A/NP for a 24-nt RNA is lower at 150 and 300 mM NaCl, suggesting that the RNA binds to another site, either on the same protomer or on a neighbour protomer. For our fluorescence anisotropy experiments, we used 6-fluorescein amidite (FAM)-labelled RNAs. By using a (UC)6-FAM3&prime; RNA with 150 mM NaCl, we observed an interesting phenomenon that gives macromolecular complexes similar to the ribonucleoprotein particles purified from the viruses.